Process of killing bacteria in pres



Patented Aug. 23, 1949 UNITED PROCESS OF KILLING BACTERIA IN PRES- ENCE OF A FATTY ACID SOAP WITH QUATERNARY COMPOUNDS Henry 0. Marks, Glen Ridge, N. 1., assignor to Wallace & Tiernan Products, Inc., a corporation of New Jersey No Drawing. Application January 19, 1945, Serial No. 573.644

This invention relates to an antiseptic composition and the method of killing or preventing growth of micro-organisms with the composition. More particularly the invention relates to a composition including an antiseptic quaternary ammoniumsalt and a salt of a bivalent metal.

The germicidal properties of quaternary ammonium salts giving a surface active cation when dissolved in water has been recognized for many years; Their held of usefulness has been limited, however. by the fact that such salts are ineffective as bactericides or bacteristats in the presence of fatty acid soaps. that is, the alkali metal salts of the higher fatty acids. The limitation is very important since there is, always the possibility that any given batch of germicide will be applied in use to a surface that has been washed with soap or may even be used in conjunction with soap, as in dish washing in a public restau-a, rant.

The present invention provides a quaternary ammonium composition and method that are effective even in the presence of such proportions of fatty acid soaps as encountered on surfaces previously washed with soap solution, It provides a composition and method that are useful in disinfecting in general, as, for instance, in sterilizing surgical instruments, washing dishes and food handling utensils, and preparing the skin of a patient and the hands of the surgeon before an operation.

Briefly stated, the invention comprises an antiseptic composition including a normally antiseptic quaternary ammonium salt, a salt of a bivalent metal, and suitably also a liquid, each of the two salts selected being soluble to a substantial extent in water or other liquid used originally or encountered on application of the composition to the micro-organisms and the two salts, when mixed together, giving no precipitate at the concentrations required for an antiseptic solution of the quaternary salt. In a preferred embodiment, both the quaternary ammonium salt and the selected bivalent metal salt are nontoxic in the proportions used. In one embodiment, the composition includes not only the two salts of the kind described but also a buffer to establish the pH at a level of at least 6 and for best results within the range 7 to 8 and ordinarily about 7.6. The invention includes also the method of killing or preventing multiplication of 1 Claim. (C1. 1.67-22) monium salt and the metal salt. The composition may be mixed dry and then the whole dissolved in water in predetermined proportion before use. In an alternative procedure, the ingredients may be added separately to the water. In another practice of the invention, the quaternary ammonium salt and the bivalent metal salt may be applied in the form of separate solutions to the surface on which the bacteria or other micro-organisms are to be killed.

The previous use of soap on such surfaces does not destroy the effectiveness of the antiseptic quaternary ammonium salt so long as there is present a large proportion of the bivalent metal salt.

Also the order of contact of the quaternary ammonium salt, the bivalent metal salt, and the fatty acid soap with the organisms may be varied. So long as the quaternary salt and the metal salt are both present finally, the order of contact of these materials and of the normally disturbing soap with the organisms is relatively unimportant. In cases in which application of the quaternary ammonium salt on the'site containing some soap has been found not to kill the bacteria present, substantially complete killing results when the bivalent metal salt is subsemust be selected one that when dissolved in soap free water gives a surface active antiseptic cation, is soluble in water at least to the concentration required to make the aqueous solution antiseptic, that is, either bactericidal or bacteristatic, is preferably non-toxic in theproportion used, particularly if the final composition is to be used on open wounds, and at the concentration required for the antiseptic solution does not give a precipitate withthe multivalent metal salt. Ex-

amples of such quaternary ammonium salts that may be used are 4-chlorocymoxyethoxyethyl 4- chlorobenzyl dimethyl ammonium halide and preferably the chloride, a long-chain-alkyl dimethyl benzyl ammonium chloride, cetyl pyridinium chloride. and p-tertiaryoctyl-phenoxyethoxy-ethyl dimethyl benzyl ammonium chloride. The quaternary compound selected is a wetting agent because of the presence of the surface active cation referred to above. In general, the quaternary ammonium compound may be one that meets the general requirements stated and is selected from those described in U. 8. Patent 2,108,765, issued on February 15, 1938, to Domagk and U. S. Patent 2,336,465, issued on December 14, 1943, to Buck et al.

A type formula for the quaternary ammonium compounds that are satisfactory provided they meet the general requirements stated is given below.

Quaternary ammonium compounds that are particularly effective as antiseptics usually have a long-chain-alkyl containing, for instance, at

C C Eva Clair- The pyridine group, holding the N with 3 valences, corresponds to all R's, in the type formula first given, except the R represented here by the cetyl group (CmHsa). In place of the benzyl group referred to above, there may be used another aralkyl radical, as, for instance, phenylethyl disclosed in the said Domagk patent or one of the substituted cymoxy (substituted benzyl) groups of Examples 1 to 6 hereof. In any case, the R's are hydrocarbon groups that may or may not be substituted or interrupted and X is an anionic component that gives with the quaternary ammonium compound a'salt that is at least appreciably soluble in water. X is usually one of the halogen atoms, preferably chlorine, although it may be some other element or group, as, for instance, acetate (OOC.CH3), propionate (OOC.C2H5), or in some cases sulfate (S04).

As the metal salt, there is used one that is bivalent and that does not cause precipitation with the quaternary ammonium salt, in the minimum concentration of the latter required to make an antiseptic solution in the absence of a fatty acid soap, when the metal salt is present also in about the same proportion as the quarternary salt. Thus, there may be used salts of the alkaline earth metals, preferably with chlorine or acetate, lead acetate, zinc chloride, zinc acetate and the like. Calcium nitrate may be used with those quaternary compounds whose nitrates are soluble.

The metals that cause precipitation of the quaternary ammonium salts even at very low concentrations are not satisfactory. Also unsatisfactory for use when the composition is to be long exposed to air before use are metals that are readily oxidizable by the atmosphere including, for instance, salts of iron, manganese, and

clude a buffer ii this is necessary to establish the pH at a level of at least 6 or at the preferred value of about 7.6., This is particularly important since the composition is more effective in establishing antisepsis under conditions that are otherwise comparable when the pH is at the levels described.

The buifer when required may be of kind that is usual for use with the quaternary ammonium salt or the bivalent metal salt separately and should precipitate no large proportion of either of them and preferably none of them. Buffers that may be used and that illustrate the class are sodium acetate, potassium acetate, sodium propionate, and calcium acetate, the latter used in adequate proportion serving well as both the bivalent metal salt and the buffer. The buffer should contain as the anionic part an element or group that does not form an insoluble salt with the quaternary ammonium cation or the bivalent metal present in the aqueous solution of the antiseptic composition, at the concentrations in which the three materials are used.

As to proportions, the quaternary ammonium salt, dissolved in water at or before the time of application to the bacteria to be killed, is used in proportion to form a solution of concentration at least as great as that known from previous experience or determined by simple test to be effective in the absence of a fatty acid soap. Thus, the quaternary ammonium salt is ordinarily used in the proportion of at least 15 and preferably 30 parts to 1,000,000 parts of water, best in the proportion of at least 50 to 300 parts per million of water (P. P. M.) when actually in contact with the micro-organisms to be killed. Since there is frequently dilution of the antiseptic solution by water or other fluid on the surface to be treated and because of the desirability of a safety factor in the form of an excess of the antiseptic, it is best to apply the antiseptic quaternary ammonium salt in the proportion of about 100 to 4,000 P. P. M. of its solution.

The proportion of the bivalent metal salt to be used is important and must be at least approximately equal to the proportion of the quaternary ammonium salt. At low concentration of the latter within the range given above,

the proportion of the bivalent metal salt should be materially larger. In fact, it is best at all concentrations, in order to obtain the most effective composition for a given concentration of the quaternary ammonium salt used, to have present a large excess of the bivalent metal compound. Thus, with proportion of the quaternary salt ranging from 100 to 4,000 P. P. M., there are I used to advantage 100 to 8,000 P. P. M. of calcium chloride. In general, the proportion of the alkali metal salt is usually about 2 to 10 parts for 1 of the quaternary ammonium salt, when calcium is the bivalent metal present. when other bivalent metals are present in the salt, the proportion should be somewhat increased as these other salts are for the most part less effective per unit of weight than the calcium salts. Thus, tests made with magnesium and zinc salts show that hile these salts are efl'ectlve for the present purpose, their proportion to the quaternary ammonium salt should be materially larger by say 2 to 10 times than when the calcium salt is used.

In any case, the proportion of the metal salt preferred is greatly in excess of that equivalent to either the quaternary ammonium salt or the fatty acid soap encountered in the use of the quaternary'salt as an antiseptic. Thus, careful tests have shown that the proportion of the metal salt should be at least approximately equal in weight to that of the quaternary ammonium salt. In most cases the proportion of metal salt should be at least approximately equal to that of the soap encountered in the use of the quaternary salt, in those cases in which the proportion of the soap is greater than that of the quaternam compound. Since a soap, consisting chiefly of a usual mixture of the sodium salts or stearic, oleic and palmitic acids, has a molecular weight (also equivalent weight) of about 300 or slightly more and since the equivalent weight of calcium chloride, for instance,,is 55.5, it will be seen that the use of equal proportions of calcium chloride and fatty acid soap represents at least 5 equivalents of the calcium salt to 1 of the fatty acid soap.

The buffer is used in proportions determined by simple test to give the desiredpI-I to the fln= ished composition when dissolved in water for use as a bactericidal solution. Usually the buflcr' if required at all would be used in the proportion of about 20% to 1.00% 01' the quaternary salt.

Since calcium chloride is inexpensive and nontoxic in moderate proportions, the efiect of the metal salt and of the proportion of it will be illustrated largely by description in connection with the use oi calcium chloride as the bivalent Table II shows the concentrations of sodium soap permitting substantially complete killing of Staphylococcus aureusby the combination or 1 6 not in 5 minutes, with each of two different quaternary ammonium compounds used in concentration of P. P. M. and with various proportions of calcium chloride, at a pH of 7.

The next test was the disinfection of hands. A number of persons (operators) washed their hands for 1 minute in each of 5 basins containin a liter of 0.05% soap solution in water, to

stabilize the rate of removal of bacteria during the rinsing. Washes'3 to 5 inclusive were collected and a bacterial count run, the count being designated in Table III as the first count.

The hands of the operators were then rinsed in plain water and then for 3 minutes with 50 cc. of a 1:100!) solution of -chlorocymoxyethoxyethyl 4-chlorobenzyl dimethyl ammonium chloride, the solution containing in some cases 10 parts of calcium chloride to 1 of the said compound i. e. 1% of the chloride on the weight of the solution) and in other cases no bivalent metal salt.

After this treatment the hands were-given three more washings with a literof 0.05% soap solution, the last three washings being collected and the bacterial count again determined as above and designated as the second count in Table III.

Table III.--Hand disinfection with the quaternary compound Bacteriaaslhou- CaCl: San Reduc- Soap Used per tion,

cent 1st 2d percent Count Count A. B Non 0 290 3 99. 2 A. B .-c.o 1 270 l 99. 8 I S r n 0 1, 400 5 99. 8 K. G rn 1 3,666 120 97. 2 O. W"--- OnlyinZd series 0 2,100 l, 600 13.8 A. 13.. (in l 640 200 69 F. S Throughout 0 l, 900 1, 600 16 O. W do 1 l, 680 B4 75 1.13 .-do 0 4,000 0,000 25 M. R. dn 1 640 58 91 1 I. e., only after treatment with the quaternary composition.

It will be seen from Table III that the reduction in the bacteria removed from the hands was metal salt. Table I shows the results of contacting Staphylococcus aureus with solutions or the germicidal compositions of the kind described 50 at a pH of 7.5. It shows for comparison the onset of compositions that are the same except for the omission of the metal salt.

Table I No. of Organisms per cc.

0 'al 1 gu ii t iy l l ien zgl Soap CaCla, Ammonium c010- P.'P. it. P. P.'M.

ride, P. P. M. 5 minutes 10 minutes very large when either (1) no soap was used on the hands of the operator in advance oi the application of the quaternary compound or (2) when the calcium chloride as well as the soap was used.

Finally, Table IV shows a series of experiments designed to'test the eflicacy or the combination in skin sterilization. Rabbit skin was used, the method employed being the one given by Habs and Kirschner (Zeit. Hygeine Infek. 124, 557, 1942) Test areas on the shaved bellies of the rah-'- bits were infected with Pseudomonas aeruglnosa. In some cases, the areas were treated with 0.1 ml.

quaternary saltland metal salt in 10 minutes but 10 o! a 0.5% soap solution. In dthers, the scan was auaseo omitted. Then those arees'which were to receive antiseptic treatment were given two applications Table IV.-Rabbit skin disinfection uaternary Positive ompound C801 Cultures Percent Percent 0 8 out of 8 0 0 present 8 out ol 8 0. 0 0 0 out of 8 0. 5 0 present 8 out cl 8 0. 5 l 0 1 out of 8 0. 5 1 present 2 out 0! 8 The invention will be further illustrated by description in connection with specific germicidal compositions made in accordance with the present invention.

In each of the following examples the first compound named is the quaternary ammonium salt and the second is the bivalent metal salt which preserves the effectiveness of the quaternary salt even in the presence of soap and, in those'cases using calcium acetate, the acetate servesalso as the buffer. In each instance, unless specifically specified to the contrary, water may be added as solvent and the percentages represent parts of the chemicals which when water is addedfare dissolved in sufllcient water to make 100 parts of the aqueous solution.

Example 1 p C h cent phloro-cymoxy-et cry-ethyl Chloro-benzyldimethyl ammonium chloride alcium chloride. 1

Example 2 p Chi h 1 cent poro-cymoxy-et oxy-et y Ghloro-benzyl dim thyl ammonium chloride"; alciumchloride. 1

Example 3 cent Chloro-cymoxy-ethox'y-ethyl gChloro-benzyldimethylammonlumchloride. Calcium acetate monohydrate 1.6

Example 4 If the composition is to ,be used in cases where a high salt concentration is not detrimental, it is desirable to increase the concentration of both If it is desired to use this concentration of wetting agent, for, example, on body fluids where excessive salt concentrations should be avoided, the calcium acetate is reduced to 1 to 2% Example '5 It is often desirable to make up concentrated solutions for purposes of storage and shipment which can'be diluted just before use. This and the following example illustrate such compositions.

Perp-om m 1 cent oro-cymor" v-e y g-Chloro-benzyl'dimethyl ammonium chloride 5 alcium monohydmm 20 Example 6 p-Chl th 1 cent oro-cymoxy -e Ohloro-benryl dlmethyl anzmonium chloride 3 alcium acetate monohydrate.- 13

The use of calcium acetate, not containing a common ion with the quaternary salt, permits of high concentration of the salt without causing precipitation by salting out.

The approximate limits to these concentrations have been investigated experimentally and it has been found, for example, that a calcium acetate concentration of 23% limits the solubility of the quaternary compound of this example to 1.8%. Decreasing the calcium acetate concentration to 15%, increases the solubility of the quaternary compound to 3.5%.

Example 7 When it is desired to make up solid mixtures to be added to water, to make solutions as needed, the following composition is suitable:

cm th in 1 'cm iztwifi fimifii aniincnium chloride.

alcium acetate monohydrate. Water, none. A

The two powders are mixed and ground up together to give auniform composition.

As indicated briefly in Example '6 above, it is sometimes necessary, when high concentrationsare desired without causing precipitation, to avoid the use of a common ion in the bivalent metal salt and quaternary ammonium salt. This is to avoid the salting out phenomenon which is sometimes observed when two materials in solu- I tion give a common ion. Calcium acetate is a desirable salt to use when the quaternary ammonium compound includes chloride, the two giving no common ionand therefore no saltins out efiect. Even when the quaternary ammonium salt is the acetate, there is ordinarily no salting out eilect from the use of calcium acetate or other bivalent metal acetate. I

In place of the aqueous solutions used above there may be .substituted solutions in other solvents ior the antiseptic composition that are miscible with water so that the germicidal composition will establish good contact with moist skin or other moist surface to be disinfected. Thus, there may be used solutions in the lower alcohols, particularly in such alcohols mixed with water. Thus there may be used aqueous solutions of ethyl, propyl, or isopropyl alcohol. The alcohol and water solutions may be substituted for an equal weight of water in the above examples. It will be understood also that it is intended to cover all changes and modifications of the examples of the invention herein chosen for the purpose of illustration which do not constitute departures from the spirit and scope of the invention.

What is claimed is:

The method of killing bacteria in the presence of a fatty acid soap which comprises contactin the bacteria and the said soap with an aqueous solution of an appreciably water soluble quaternary ammonium salt that when dissolved in water gives a surface active antiseptic cation and is of the general formula HENRY C. MARKS.

10 REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,152,047 Hahl et al Mar. 28, 1939 2,190,133 Epstein et a1. Feb. 13, 1940 10 2,295,504 Shelton Sept. 8, 1942 2,295,505 Shelton Sept. 8, 1942 OTHER REFERENCES Stitt, Ann. Otol. Rhin. & Laryng., 52, 4'77 (June 1943), thru Squibb Abstr. 31111., vol. 16, page 15 1226, August 18, 1943. 

